WO1993022423A1 - Phospholipides nutritifs pour bacteries pathogenes - Google Patents

Phospholipides nutritifs pour bacteries pathogenes Download PDF

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Publication number
WO1993022423A1
WO1993022423A1 PCT/US1993/004053 US9304053W WO9322423A1 WO 1993022423 A1 WO1993022423 A1 WO 1993022423A1 US 9304053 W US9304053 W US 9304053W WO 9322423 A1 WO9322423 A1 WO 9322423A1
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composition
bacterial cells
mucus
lipids
phosphatidylserine
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PCT/US1993/004053
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English (en)
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Howard C. Krivan
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Microcarb Inc.
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Publication of WO1993022423A1 publication Critical patent/WO1993022423A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor

Definitions

  • the present invention relates generally to bacteria, including pathogenic bacteria, and- their growth on lipids.
  • This invention is more particularly related to methods for growing bacterial cells, the production of mutant strains which cannot grow in animals and their use as host cells for expression of cloned DNA molecules, bacterial cells expressing proteins induced or enhanced by growth on lipids, and the use of such cells, fractions thereof, or individual proteins in vaccines.
  • Mucosal surfaces in various locations throughout the body have been suggested to serve as barriers to bacterial infection as well as sites for bacterial colonization.
  • the large and small intestinal walls consist of an epithelium containing brush border epithelial cells and goblet cells which secrete a relatively thick (up to 400 ⁇ m) viscous, mucus covering.
  • the epithelial cells synthesize glycoproteins and glycolipids which are integrated into the brush border membranes, thereby forming the epithelial cell glycocalyx.
  • the mucus layer contains mucin, a 2 x 10 6 dalton gel- forming glycoprotein, and a large number of smaller components.
  • shed epithelial cells are the source of many of the smaller components of mucus.
  • the intestinal mucus layer itself is in a dynamic state, continuously being synthesized and secreted by goblet cells and degraded to a large extent by intestinal indigenous microflora. Degraded mucus components are shed into the lumen of the intestine and eventually find their way into feces.
  • the present invention provides a variety of methods and molecules related to bacteria, including pathogenic bacteria such as enteric bacteria and enteric invasive bacteria.
  • methods are provided for g.-.owing bacteria through the use of a variety of compositions and substances.
  • the composition consists of lipids, acidic lipids or phospholipids, each of which includes phosphatidylserine.
  • the composition comprises mucus, egg, or milk substantially free of proteins normally associated with the mucus, egg or milk, respectively.
  • the composition comprises mucus, egg or milk lipids substantially free of proteins normally associated with the mucus, egg or milk, respectively, or consists of mucus, egg or milk lipids.
  • the composition comprises mucus, egg or milk acidic lipids substantially free of proteins normally associated with the mucus, egg or milk, respectively, or consists of mucus, egg or milk acidic lipids.
  • the composition comprises mucus, egg or milk phospholipids substantially free of proteins normally associated with the mucus, egg or milk, respectively, or consists of mucus, egg or milk phospholipids.
  • the substance consists of phosphatidylserine, which may be derived from mucus, egg or milk.
  • methods are provided for selecting for a mutant strain of a bacteria. The methods comprise: exposing bacterial cells to phosphatidylserine, or to a composition consisting of lipids, acidic lipids or phospholipids, the composition including phosphatidylserine, or to a composition comprising mucus, mucus lipids, mucus acidic lipids or mucus phospholipids, the composition substantially free of proteins normally associated with the mucus, or to a composition comprising egg, egg lipids, egg acidic lipids or egg phospholipids, the composition substantially free of proteins normally associated with the egg, or to a composition comprising milk, milk lipids, milk acidic lipids or milk phospholipids, the composition substantially free of proteins normally associated with the milk, or to a composition consisting of mucus lipids
  • isolated mutant strains of bacteria produced by the methods of the present invention may be used to express a cloned DNA molecule introduced into the bacteria.
  • a method for isolating bacterial proteins whose expression are induced or enhanced by growth in the presence of phosphatidylserine or a composition including phosphatidylserine comprises: (a) growing bacteria in the presence of phosphatidylserine or a composition including phosphatidylserine under conditions and for a time sufficient to promote growth; (b) separating the proteins of the bacteria; (c) growing control bacteria under conditions and for a time sufficient to promote growth, the control bacteria growing in media in the absence of phosphatidylserine or the composition; (d) separating the proteins of the control bacteria; (e) comparing the proteins separated in steps (b) and (d) ; and (f) isolating a protein ' from the bacteria, the protein absent from the control bacteria or present in lower amount in the control bacteria.
  • Such proteins may be used as diagnostic markers to detect bacteria, especially pathogenic bacteria.
  • Such proteins may also be used in a vaccine comprising
  • the present invention provides methods for preparing bacteria or fractions thereof for use within a vaccine.
  • the method comprises: (a) growing bacteria in the presence of phosphatidylserine or a composition including phosphatidylserine under conditions and for a time sufficient to promote growth; and (b) isolating the bacteria.
  • the method comprises:
  • the method comprises: (a) growing bacteria in the presence of phosphatidylserine or a composition including phosphatidylserine under conditions and for a time sufficient to promote growth; and (b) isolating the outer membranes from the bacteria.
  • the method comprises: (a) growing bacteria in the presence of phosphatidylserine or a composition including phosphatidylserine under conditions and for a time sufficient to promote growth; and
  • Figure 1 graphically depicts the results of high-performance liquid chromatography of hydrolytically released serine from phosphatidylserine.
  • Standard phosphatidylserine, 1 g (Panel a) , and the acidic lipid fraction of mouse cecal mucus, 20 ⁇ g (Panel b) were hydrolyzed and derivatized with phenylisothiocyanate, and injected on a Supelcosil LC-18 (250 mm x 4.6 mm) column.
  • the column was eluted with a linear gradient of ammonium acetate-trimethylamine as described by Bidlingmeyer et al. (J. Chromatocrr. 116:93-104, 1984) using a- UV detector operated at 254 nm.
  • Figure 2 pictorially depicts a comparison of proteins from the outer membrane and periplasm of Salmonella typhimurium or E . coli F-18 grown in L-broth or mucus dialysate.
  • Panel A Outer Membrane and Panel B - Periplasm. 100,000 cpm were added to each lane.
  • Std protein standards
  • L-Ec L-broth grown E. coli F-18
  • L-St L-broth grown 85. tvphimurium
  • D-Ec mucus dialysate grown E. coli F-18
  • D-St mucus dialysate grown S.. tvphimurium.
  • Figure 3 pictorially depicts a comparison of proteins from periplasm of Campylobacter ieiuni grown in mucus or Brucella broth.
  • Lane 1 - C. eiuni periplasm from rabbit mucus grown cells probed with rabbit antiserum against rabbit mucus grown cells.
  • Lane 2 - C. ieiuni periplasm from Brucella broth grown cells probed with rabbit antiserum against rabbit mucus grown cells.
  • mutant strains incapable of colonization may be selected for and molecules specific for colonization may be isolated.
  • Such mutant strains and colonization-specific molecules have a variety of uses including as host cells with improved safety profiles for expression of a cloned DNA molecule and as a vaccine, respectively.
  • the present invention is directed toward methods for growing bacterial cells and methods for selecting for a mutant strain of a bacterium which is incapable of growing on mucus. Such a mutant is incapable of colonization and/or is avirulent.
  • mutant bacterium may be ' used for expression of a cloned DNA molecule which has been introduced into the bacterium. Mutant strains such as these would also be safe to be released into the environment.
  • the present invention is also directed toward bacteria containing proteins whose expression has been induced or enhanced, and the use of such bacteria, fractions thereof or proteins in vaccines and as diagnostic markers.
  • Bacteria which utilize lipids in mucus for growth include pathogenic bacteria, i.e., bacteria which are capable of producing pathological change or disease. Examples of pathogenic bacteria include Pseudomonas. Streptococcus, Staphylococcus, E. coli, Haemophilus. Mvcobacterium. Proteus. Klebsiella, Neisseria. Branhamella, Bacteroides, Listeria, Enterococci. Vibrio. Yersinia.
  • enteric bacteria Bacteria which reside in a large or small intestine or a stomach are enteric bacteria and may be invasive or non-invasive.
  • enteric invasive bacteria include Salmonella (such as S_. typhimurium and S_. cholera-suis) , Yersinia (such as Y. entercolytica) , Shigella (such as S_. dysenteriae) , Ca pylobacter (such as C. ieiuni) , and Helicobacter (such as H. pylori) .
  • enteric invasive bacteria include Salmonella (such as S_. typhimurium and S_. cholera-suis) , Yersinia (such as Y. entercolytica) , Shigella (such as S_. dysenteriae) , Ca pylobacter (such as C. ieiuni) , and Helicobacter (such as H. pylori) .
  • pathogenic bacteria are preferred
  • the present invention provides methods for growing bacterial cells through the use of a variety of compositions and substances.
  • the composition comprises mucus substantially free of protein normally associated with the mucus.
  • Types of mucus include intestinal, gastric, and respiratory mucus.
  • a mucus sample may be obtained from a variety of sources, including mammals such as humans, rabbits or mice, or birds such as chickens.
  • mucus may be prepared from mouse intestines. Briefly, animals, whose intake for the preceding 24 hours is only sterile water containing antibiotic, are sacrificed and the small intestines removed.
  • the mucus layer covering the mucosal surface is isolated (e.g., by scraping with a rubber spatula).
  • the mucosal scrapings are separated from particulate and cellular material (e.g., by centrifugation) to yield mucus in the supernate.
  • Substantially all of the proteins residing in a mucus sample may be generally removed by extraction(s) using salt, detergent and/or organic solvents. Briefly, for example, removal may be accomplished by extraction with chloroform/ir-ethanol (2:1) to yield mucus substantially free of proteins normally associated with mucus.
  • mucin a protein normally associated with mucus
  • mucin a 2 x 10 6 dalton gel- forming glycoprotein.
  • the composition of the mucus which remains following treatment of the mucus to remove proteins should include lipids.
  • the presence of lipids may be verified by well-known analytical techniques, such as chromatography (e.g., thin layer or high-performance liquid chromatography) .
  • the composition comprises mucus lipids substantially free of proteins normally associated with the mucus.
  • total lipids may be separated from mucus by organic solvent extraction of mucus.
  • separation may be accomplished by extraction of mucus (dialyzed against water) using chloroform/methanol/water (e.g., 4:8:3) or chloroform/methanol (e.g., 2:1).
  • a lipid fraction typically includes acidic lipids, neutral lipids, triglycerides, fatty acids, and glycolipids.
  • the composition comprises mucus acidic lipids substantially free of proteins normally associated with the mucus.
  • acidic lipids may be separated from mucus by chromatography, such as high-performance chromatography and ion exchange chromatography.
  • separation may be accomplished by high-performance chromatography on silica Iatrobeads and ion exchange chromatography on DEAE cellulose.
  • Acidic lipid fractions include phospholipids, sulfatides, and gangliosides.
  • the composition comprises mucus phospholipids substantially free of proteins normally associated with the mucus.
  • phospholipids may be separated from mucus lipids by extraction or chromatography. For example, separation may be accomplished by ion exchange chromatography.
  • a phospholipid fraction includes phosphatidylserine.
  • compositions described above may be desirable to add one or more substances to the compositions described above, e.g., for the purpose of enhancing growth.
  • one or more amino acids or proteins which are not normally associated with the mucus may be added to any of the compositions which are substantially free of proteins normally associated with the mucus.
  • other nutrients or salts may be added to any of the compositions.
  • substances suitable for use in methods for growing bacterial cells include individual phospholipids such as phosphatidylserine.
  • mucus phosphatidylserine may be separated from mucus phospholipids by chromatography. For example, separation may be accomplished by ion exchange chromatography.
  • the disclosure of the present invention shows that bacteria utilize phosphatidylserine in mucus for growth.
  • lipid fractions or subtractions from mucus include other sources (such as avian egg or milk) for naturally derived lipid fractions or subfractions, the synthesis (e.g., chemically and/or enzymatically) of lipids for use within a composition as described above, or preparation of a composition using commercially available lipids (e.g., phospholipids are available from Avanti Polar Lipids, Inc., Alabaster, Alabama) , or preparation of a composition using a combination of sources.
  • sources such as avian egg or milk
  • synthesis e.g., chemically and/or enzymatically
  • Lipid fractions may be extracted from sources other than mucus, such as eggs, egg yolk extract (commercially available, e.g., from Miles Laboratories) , or lyophilized milk, using the procedures described above for mucus. Acidic lipids and phospholipids may be separated from such lipid fractions using the procedures described above for mucus. It is desirable that the lipid fraction or subtraction, whether naturally derived or synthetically prepared, include phosphatidylserine. Briefly, phospholipids may be synthesized using glycol analogs (e.g., H. Eibl, "Synthesis of Glycerophospholipids, " Chemistry and Physics of Lipids 26:405-429, 1980) .
  • glycol analogs e.g., H. Eibl, "Synthesis of Glycerophospholipids, " Chemistry and Physics of Lipids 26:405-429, 1980.
  • oleoxylpropandiol-(1,3) can be combined with phosphorous oxychloride and reacted with N-t-butyloxy-carbonyl-L- serine phthalimidomethylester in the presence of pyridine.
  • the protecting groups are then removed by hydrazonolysis and treatment with formic acid.
  • phosphatidylserine e.g., derived from bovine brain
  • one or a combination of synthetic phosphatidylserines such as those with fatty acid chains containing C6, C8, C12 and C18 may be used.
  • bacterial cells are exposed to one or more of the compositions or substances described above. It will be evident to those of ordinary skill in the art that there are a variety of ways of exposing or contacting cells with a composition or a substance. For example, bacterial cells may be incubated with a composition or a substance.
  • lipid fractions or purified lipid are dried under nitrogen, dispersed by sonication into HEPES (N-2- hydroxyethylpiperazine-N' -2-ethanesulfonic acid) -Hanks buffer (pH 7.4) at 1 mg/ml, and inoculated with about 2 x 10 4 colony forming units ("CFU") of bacteria per ml. Incubation takes place under conditions and for a time sufficient to permit growth of the bacterial cells. For example, cells may be incubated with a composition or a substance for 6 hours at 37°C. Growth of the bacterial cells may be determined and monitored qualitatively (e.g., by visualization or microscopic examination) or quantified (e.g., by plate counts) .
  • compositions or substances may be prepared in the form of plates or broth to be used as bacteriological media within the methods of the present invention for growing bacterial cells.
  • agar NOBL Denssion Laboratories, Detroit, MI
  • HEPES-Hanks buffer pH 7.4
  • a substance such as phosphatidylserine (10-20 mg/ml) or a composition such as mucus lipids (5-10 mg/ml) is added and the plates are gently rotated until the agar solidifies.
  • the plates may be stored at 4°C until used.
  • 1% glucose can be substituted for the carbon source and 1 mg/ml of lipid added to supply the nitrogen source.
  • lipid For broths, for example, sterile lipid (5-25 mg/ml) is dispersed in HEPES-Hanks buffer (pH 7.4), containing 1-5 mg/ml of Brij 58.
  • HEPES-Hanks buffer pH 7.4
  • Such media is useful for culture, propagation and for culturing bacteria for antimicrobial testing.
  • methods are provided for selecting for a "mutant strain" of a bacterium.
  • mutant strain refers to a bacterial strain which is unable to grow in mucus and incapable of intestinal or host colonization, whereas other strains are capable of growth in mucus.
  • the methods comprise exposing bacterial cells to one or more of the compositions or substances described above and selecting for a mutant strain. Mutants of interest are unable to grow in the presence of phosphatidylserine as the sole source of carbon and nitrogen but continue to be able to grow utilizing D-glucose as the sole carbon source and ammonium chloride as the sole source of nitrogen. Briefly, for example, E.
  • coli strains are mutagenized with pUJIO, a suicide plasmid, which contains a 3-lactamase gene external to a TnphoA mobile element and which carries the transposase gene in cis but also external to the TnphoA mobile element (see, for example, de Lorenzo et al., J. Bacteriol. 172:6568, 1990).
  • the TnpJigA mobile element also contains a neomycin phosphotransferase gene, conferring kanamycin resistance. Since the transposase is physically separated from the transposable element of this vector, it is lost as the vector is lost, thereby resulting in stable transposition and in the isolation of stable mutants resistant to kanamycin but sensitive to ampicillin.
  • Mutants are first screened on agar plates for the ability to grow utilizing glucose and ammonium chloride as the sole source of carbon and nitrogen. Those that are able to do so are tested, using 96 well polystyrene plates, for the ability to grow using phosphatidylserine as the sole source of carbon and nitrogen. Mutants that are unable to utilize phosphatidylserine for growth are tested for the ability to grow in mouse cecal mucus in vitro and to colonize the intestines of mice in vivo. Those that fail to grow in mouse cecal mucus and fail to colonize may be tested for the ability to grow in human colonic mucus in vitro. Mutants that fail to grow in human mucus are preferred strains.
  • mutant strains of the present invention have a variety of uses.
  • mutant strains may be used as host cells for expression of a cloned DNA molecule which has been introduced into the mutant bacterium.
  • a mutant strain must be capable of expressing the cloned DNA molecule introduced into it. Expression capability may be confirmed by, for example, transforming random sequences of DNA isolated from the wild-type strains into a sample of cells of a mutant strain. Mutants containing sequences expressing the lipid transport proteins can be identified as now being able to grow utilizing lipids or phosphatidylserine as the sole source of carbon and nitrogen.
  • the recombinant plasmids may be extracted, transformed into a fresh transport mutant background, and shown to gain the ability to utilize the lipids and phosphatidylserine for growth.
  • a sequence once a sequence is identified it can be subcloned and placed in the appropriate expression vector for isolation of large quantities of the protein.
  • An expression vector may be constructed and then used to transform a microorganism for the expression and production of a protein.
  • recombinant plasmids capable of integration into a host mutant cell comprise a promoter followed downstream by a DNA sequence encoding a protein. It may be desirable to include a polyadenylation signal downstream from the DNA sequence.
  • One embodiment of a method for producing a protein comprises introducing into a host mutant cell a DNA sequence encoding a protein. The host cells are grown in an appropriate medium and the protein product encoded by the DNA sequence produced by the host cell is isolated. Examples of techniques known in the art include those disclosed in U.S.
  • the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • an expression vector such as a plasmid
  • the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Not all of the hosts may be transformed by the vector. Therefore, it may be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, -that codes for a selectable trait in the transformed cell, such as antibiotic resistance.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Mutant host cells of the present invention express protein(s) encoded by a cloned DNA molecule(s) which has been introduced into the mutant bacterium, but are unable to grow on mucus. Such cells are cultured by known techniques, and the proteins are recovered by known techniques. Depending upon the expression system used, the recombinant proteins expressed may be part of a fusion protein produced by the transformed host cells. Such proteins are recovered by known techniques, and the undesired part may be removed by known techniques. Alternatively, the fusion protein itself may be more immunogenic than the recombinant protein or polypeptide alone and, therefore, may itself be useful, e.g., in a vaccine.
  • Such mutant strains would be desirable hosts for recombinant DNA research as they grow well in laboratory media, but are unable to grow in mucus and colonize. Thus, these mutants would provide new cloning vectors with improved safety profiles for introduction into the environment.
  • a Salmonella or Pseudomonas mutant of the present invention is environmentally safe for release if it is non-invasive because of its inability to grow on host mucus and phosphatidylserine.
  • the present invention provides methods for identifying or isolating bacterial proteins whose expression is induced, or at least enhanced, by growth under conditions which are associated with colonization. Certain proteins are either not expressed or only expressed in low levels under standard laboratory culture conditions.
  • One method for identifying and isolating such proteins in substantially pure form is to first compare (e.g., by sodium dodecyl sulfate- polyacrylamide gel electrophoresis) the proteins produced by a bacterium which is capable of utilizing phosphatidylserine for growth and which is grown in the presence of phosphatidylserine (i.e., grown on phosphatidylserine or on a composition including phosphatidylserine) , with the proteins produced by the bacterium when grown in the absence of phosphatidylserine ("control") .
  • proteins may be identified which are present in the bacterium grown in the presence of phosphatidylserine but which are absent in the control bacterium, or at least present in the control bacterium in lower levels.
  • a protein with a molecular mass of 45-46 kdal is expressed in the outer membranes of mucus grown cells, but is expressed only minimally in L-broth grown cells.
  • Other examples of bacterial proteins whose expression is induced or enhanced, by growth of bacteria in phosphatidylserine or compositions containing phosphatidylserine include E.
  • proteins may then be isolated in substantially pure form.
  • the proteins of a bacteria or a protein fraction may be isolated from bacteria (e.g., by extraction and/or centrifugation) and separated from one another (e.g., by polyacrylamide gel electrophoresis) .
  • the same procedure is performed using control bacteria.
  • separated proteins may be detected using a variety of techniques, such as stains, antibodies and the like. Detection of proteins which are present in small quantities in even the non-control bacteria may be accomplished by additional amplification of the detection of the separated proteins, e.g., by growing the bacteria in the presence of radioactive amino acids which metabolicly label the bacteria's proteins.
  • a comparison of the separated proteins from control and non-control bacteria is made (e.g., visually, spectrophotometrically, etc.) to determine which proteins are present only in the non-control bacteria, or at least present in greater amounts in the non-control bacteria relative to the control bacteria.
  • individual proteins may be isolated by a variety of techniques well known to those of ordinary skill in the art. Such techniques include extraction and/or chromatography.
  • a substantially pure protein may be analyzed by various analytical techniques, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) .
  • proteins identified or prepared by the methods of the present invention may be used in vaccines to prevent diseases associated with pathogenic bacterial infections.
  • Proteins identified or isolated by the methods of the present invention may be prepared by other methods for use in vaccines.
  • a protein may be prepared synthetically (chemically and/or enzymatically) or by recombinant technology, using methodologies well known to those in the art. Interference with the colonization of host cells by pathogenic bacteria (such as enteric invasive bacteria) is an effective basis for the prevention of the diseases which they cause. Administration of such proteins as a vaccine leads to an immune response in which antibodies which bind to the protein are produced. These antibodies inhibit colonization of host cells by the pathogenic bacteria.
  • whole bacterial ' cells or fractions thereof, from bacteria grown using the methods of the present invention may be used in vaccines.
  • whole cell vaccines are prepared from about 1 x 10 11 bacteria which are harvested by centrifugation from media containing, for example, mucus, lipids derived from mucus, or phosphatidylserine, and washed three times with PBS. Bacteria are either inactivated by hearing at 56°C for 30 min. or by treating cells with 0.025 M formaldehyde at room temperature for 24 h and at 4°C for 24 h.
  • the vaccine is typically administered in three oral doses at 2 week intervals or injected intramuscularly three times over a three to five week interval.
  • outer membrane and/or periplasmic fractions from bacteria grown using the methods of the present invention, may be used in vaccines.
  • outer membrane and/or periplasm may be further fractionated to yield the respective protein subtractions.
  • Such collections of outer membrane proteins and/or periplasmic proteins may be used in vaccines. Briefly, bacteria are grown, for example, in media containing mucus dialysate, lipids derived from mucus or phosphatidylserine. Whole cells are washed three times in PBS and sonicated three times on ice with microtip setting at 2 for 30 s each. After sonication, the cellular debris is removed by centrifugation at 5000 x g for 20 min.
  • the inner membrane is digested with 2% Sarkosyl in 7 mM-EDTA, pH 7.6, at 37°C with gentle rocking for 30 min.
  • the suspension is centrifuged again at 100,000 x g for 2 h and the supernatant containing the periplasmic proteins and the pellet containing Sarkosyl insoluble outer membrane proteins are collected.
  • the proteins may be analyzed by SDS-PAGE. For example, samples (10 ⁇ g) are run in 10% polyacrylamide gels under reducing conditions by the standard procedure of Laemmli. Gels are stained with Coomassic blue stain or silver stain (Bio-Rad) to visualize the protein bands.
  • bacteria are grown in media containing 35 S-methionine and - 35 S-cysteine, membrane proteins are prepared as above and 100,000 cpm are added to each lane prior to SDS-PAGE analysis.
  • Outer membrane proteins may also be prepared by lithium chloride-lithium acetate extraction (e.g., as described by Johnson et al., Infection and Immunity 57:1809-1815, 1989).
  • Bacteria are suspended in buffer containing 0.2 M lithium chloride and 0.1 M lithium acetate. The pH is adjusted to 6.0 with acetic acid.
  • Membrane vesicles are generated by shaking the cell suspension at 250 rpm at 45°C for 2 h in flasks containing 3- ⁇ nm glass beads.
  • a vehicle for antigen delivery examples include aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes.
  • immunostimulatory substances include N-acetylmuramyl-L- alanine-D-isoglutamine (MDP) , lipopoly-saccharides (LPS) , and glucan.
  • a protein may be prepared synthetically and that a portion of the protein (naturally-derived or synthetic) may be used.
  • a peptide of a protein it may be desirable to couple the peptide hapten to a carrier substance, such as keyhole limpet hemocyanin.
  • the proteins of the present invention may also be used as diagnostic markers to detect bacteria, such as pathogenic bacteria.
  • phosphatidylserine may be immobilized on a solid support (such as beads) and contacted with a sample containing bacteria (e.g., in a bodily fluid such as urine) .
  • Phosphatidylserine may be immobilized onto a solid support (such as microtiter wells or chromatographic resins) by adsorption or covalent attachment. It will be evident that phosphatidylserine may be covalently attached in a variety of ways, including linker groups such as those available from Pierce Chemical Co. (Rockford, 111.) .
  • a protein whose expression in induced by phosphatidylserine, is then detected directly or indirectly.
  • antibodies which specifically bind to such a protein may be utilized.
  • Polyclonal or monoclonal antibodies (MAbs) which are capable of specifically binding (i.e., with a binding affinity of about 10° liters per mole) a protein of the present invention may be produced.
  • polyclonal antibodies may be produced by immunization of an animal with a protein and subsequent collection of its sera. Immunization is accomplished, for example, by systemic administration, such as by subcutaneous, intraplenic or intramuscular injection, into a rabbit, rat or mouse. It is generally preferred to follow the initial immunization with one or more booster immunizations prior to sera collection. Such methodology is well known and described in a number of references.
  • MAbs may be generally produced by the method of Kohler and Milstein (Nature 256.:495-497, 1975; Eur. J. Immunol. 6:511-519, 1976) . Briefly, cells of lymph nodes and/or spleens of an animal immunized with a protein are fused with myeloma cells to form hybrid cell lines ("hybridomas" or "clones") . Each hybridoma secretes a single type of immunoglobulin specific for the protein, and, like the myeloma cells, has the potential for indefinite cell division.
  • Suitable MAbs include those of murine or human origin, or chimeric antibodies such as those which combine portions of both human and murine antibodies (i.e., antigen binding region of murine antibody plus constant regions of human antibody) .
  • Human and chimeric antibodies may be produced using methods well known by those skilled in the art.
  • An alternative to the production of MAbs via hybridomas is the creation of MAb expression libraries using bacteriophage and bacteria
  • mice Five to 8-week-old CD-I mice (Charles River Breeding Laboratories, Inc., Wilmington, Mass.) . Twenty-four hours before use the mice were deprived of food and given sterile water containing 0.5% (wt/wt) streptomycin sulfate. The following day the animals (usually four to six) were sacrificed, and the small intestines were removed and placed in sterile petri dishes containing HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) -Hanks buffer (pH 7.4). The individual intestines were pooled and cut into 2- to 3-cm lengths.
  • HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
  • Any feces and partially digested food present were expressed from each section with a rubber spatula.
  • the sections were then transferred to a second set of petri dishes containing HEPES-Hanks buffer (pH 7.4) and split open with a scalpel.
  • the split sections were agitated to remove any remaining debris and transferred to a third set of petri dishes.
  • Each section was then gently scraped with a rubber spatula to remove the mucus layer covering the mucosal surface.
  • the mucosal scrapings were centrifuged at 27,000 x g for 15 minutes to remove particular and cellular material. The resulting supernates contained the mucus.
  • typhimurium SL5319 grew better than E. coli F-18 were pooled and dialyzed (Spectra/Por 3 dialysis tubing, 11.5 mm diameter, 3500 d cutoff, Los Angeles, CA) against 30 v of HEPES-Hanks buffer, pH 7.4, for 6 hours at 5°C. Dialysates were lyophilized and resuspended in their original volume. Lipids were extracted from mucus in chloroform/methanol/water (4:8:3) (Svennerhold et al., Biochim. Biophys. Acta 617:97-109. 1980) or in chloroform/methanol (2:1) (Slomiany et al., J. Biol. Chem.
  • the acidic lipid fraction was hydrolyzed with 6N HCl for 18 hours at 120°C, and released serine was measured by the PICO-TAG method using phenylisothiocyanate (Cohen et al., Nature 320:769-770,
  • E. coli 933 EDL (0157:H7) a human enterohemorragic (“EHEC”) strain
  • E. coli A55 06, pap*, hly +
  • UTI human urinary tract infection
  • Phospholipids, sulfatides and gangliosides make up the majority of acidic lipids, whereas the majority of neutral lipids are neutral glycolipids. Therefore, several phospholipids (phosphatidylcholine, phosphatidyl- ethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin; Avanti Polar Lipids, Inc., Alabaster, Alabama) , purified mixtures of standard monosialyl- gangliosides (GM ⁇ , G 2, and GM3; BioCarb Chemicals, Lund, Sweden), disialylgangliosides, (GD]_ a , GDn-,, GD2, and GD3; BioCarb Chemicals) , and a standard mixture of neutral glycolipids which contained galactosylceramide, lactosylceramide, globotriaosylceramide, globoside, and Forssman glyco
  • Salmonella and E. coli are capable of utilizing cecal mucus total lipids, cecal mucus acidic lipids, and phosphatidylserine, as the sole sources of carbon and nitrogen and that they do so without an extended lag period.
  • Lipids were dispersed in HEPES-Hanks buffer, pH 7.4 at a concentration of 1 mg per ml.
  • HEPES-Hanks buffer pH 7.4 at l mg per ml.
  • c HEPES-Hanks buffer pH 7.4.
  • Phospholipids were dispersed in HEPES-Hanks buffer, pH 7.4 at a concentration of 1 mg per ml.

Abstract

L'invention se rapporte à divers procédés permettant la croissance de cellules bactériennes sur des lipides, des lipides acides, des phospholipides, de la phosphatidylsérine, ou sur des fractions ou sous-fractions de mucus, d'÷ufs ou de lait. Das bactéries pathogènes sont de préférence produites, et comprennent des bactéries telles que Salmonella, Yersinia, Shigella, Campylobacter, Helicobacter, Pseudomonas, Streptococcus, Staphylococcus, E. coli, Haemophilus, Mycobacterium, Proteus, Klebsiella, Neisseria, Branhamella, Bacteroides, Listeria, Enterococci, Vibrio, Yersinia, Bordetella, Clostridium, Treponema et Mycoplasma. La présente invention se rapporte également à des procédés de sélection de souches mutantes qui ne peuvent être produites chez des animaux, ainsi qu'à l'utilisation de ces mutants comme cellules hôtes pour l'expression de molécules d'ADN clonées. En outre, des procédés sont décrits, lesquels permettent d'isoler des protéines dont l'expression est induite ou favorisée par la croissance en présence de phosphatidylsérine ou de compositions contenant celle-ci. De telles protéines s'adaptent à de nombreuses utilisations, y compris comme constituants de vaccins et marqueurs diagnostiques. La présente invention se rapporte également à des procédés de préparation de bactéries ou de fractions bactériennes pouvant être utilisées dans des vaccins cellulaires ou acellulaires.
PCT/US1993/004053 1992-04-29 1993-04-29 Phospholipides nutritifs pour bacteries pathogenes WO1993022423A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
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WO1994029435A1 (fr) * 1993-06-07 1994-12-22 Pharmacia Ab Expression amelioree de proteines au moyen d'un milieu de culture contenant des lipides membranaires
WO1997005899A2 (fr) * 1995-08-04 1997-02-20 University Of Guelph Nouveaux vaccins et nouvelles compositions pharmaceutiques utilisant des vesicules de membrane de micro-organismes et leurs procedes d'elaboration
US5679564A (en) * 1994-10-05 1997-10-21 Antex Biologics, Inc. Methods for producing enhanced antigenic campylobacter bacteria and vaccines
EP0900839A1 (fr) * 1997-07-25 1999-03-10 Microbiol S.n.c. Milieu de culture pour l'isolement et l'identification des Hélicobactéries, en particulier de Hélicobacter pylori
WO1999045098A2 (fr) * 1998-03-06 1999-09-10 Bruggen Pierre B V D Apport de proteines a des cellules eucaryotes au moyen de la yersinia recombinee
US6051416A (en) * 1994-10-05 2000-04-18 Antex Biologics Inc. Methods for producing enhanced antigenic Helicobacter sp.
US6916478B2 (en) 1995-08-04 2005-07-12 University Of Guelph Vaccines and pharmaceutical compositions using membrane vesicles of microorganisms, and methods for preparing same

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INFECT. IMMUN. vol. 56, no. 9, September 1988, AM.SOC.MICROBIOL.,BALTIMORE,US; pages 2209 - 2217 B.A. MCCORMICK ET AL. 'Roles of motility, chemotaxis, and penetration through and growth in intestinal mucus in the ability of an avirulent strain of Salmonella typhimurium to colonize the large intestine of streptomycin-treated mice' cited in the application *
INFECT. IMMUN. vol. 60, no. 9, September 1992, AM. SOC.MICROBIOL.,BALTIMORE, US; pages 3943 - 3946 H.C.KRIVAN ET AL. 'Phosphatidylserine found in intestinal mucus serves as a sole source of carbon and nitrogen for Salmonellae and Escherichia coli' *
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029435A1 (fr) * 1993-06-07 1994-12-22 Pharmacia Ab Expression amelioree de proteines au moyen d'un milieu de culture contenant des lipides membranaires
US5897475A (en) * 1994-10-05 1999-04-27 Antex Biologics, Inc. Vaccines comprising enhanced antigenic helicobacter spp.
US6083683A (en) * 1994-10-05 2000-07-04 Antex Biologics Inc. Methods for detecting shigella bacteria or antibodies to shigella bacteria with an immunoassay
US5679564A (en) * 1994-10-05 1997-10-21 Antex Biologics, Inc. Methods for producing enhanced antigenic campylobacter bacteria and vaccines
US5681736A (en) * 1994-10-05 1997-10-28 Antex Biologics, Inc. Methods for producing enhanced antigenic shigella bacteria and vaccines comprising same
US5869066A (en) * 1994-10-05 1999-02-09 Antex Biologics Inc. Vaccine containing a campylobacter bacterium having an enhanced antigenic property
US5858352A (en) * 1994-10-05 1999-01-12 Antex Biologics Inc. Vaccine containing a Shigella bacterium having an enhanced antigenic property
US5976525A (en) * 1994-10-05 1999-11-02 Antex Biologics Inc. Method for producing enhanced antigenic enteric bacteria
US6051416A (en) * 1994-10-05 2000-04-18 Antex Biologics Inc. Methods for producing enhanced antigenic Helicobacter sp.
US6077678A (en) * 1994-10-05 2000-06-20 Antex Biologics Inc. Methods for detecting Campylobacter bacteria or antibodies to Campylobacter bacteria with an immunoassay
WO1997005899A2 (fr) * 1995-08-04 1997-02-20 University Of Guelph Nouveaux vaccins et nouvelles compositions pharmaceutiques utilisant des vesicules de membrane de micro-organismes et leurs procedes d'elaboration
WO1997005899A3 (fr) * 1995-08-04 1997-05-29 Univ Guelph Nouveaux vaccins et nouvelles compositions pharmaceutiques utilisant des vesicules de membrane de micro-organismes et leurs procedes d'elaboration
US6916478B2 (en) 1995-08-04 2005-07-12 University Of Guelph Vaccines and pharmaceutical compositions using membrane vesicles of microorganisms, and methods for preparing same
EP0900839A1 (fr) * 1997-07-25 1999-03-10 Microbiol S.n.c. Milieu de culture pour l'isolement et l'identification des Hélicobactéries, en particulier de Hélicobacter pylori
WO1999045098A2 (fr) * 1998-03-06 1999-09-10 Bruggen Pierre B V D Apport de proteines a des cellules eucaryotes au moyen de la yersinia recombinee
US6602506B1 (en) 1998-03-06 2003-08-05 Ludwig Institute For Cancer Research Delivery of proteins into eukaryotic cells with recombinant Yersinia
WO1999045098A3 (fr) * 1998-03-06 1999-12-23 Bruggen Pierre B V D Apport de proteines a des cellules eucaryotes au moyen de la yersinia recombinee

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